Analyzing individual cells is technically more challenging compared to measuring the averaged outcome from a cell population. Such tasks are commonly performed with limiting dilution or fluorescence-activated cell sorting (FACS). Limiting dilution is based on placing diluted cell suspension in culture wells (e.g., plastic well plates) to obtain one-cell-in-a-well events, and is widely used for single cell assays such as colony formation of cancer stem/initiating cells. This method is convenient but low-throughput without using pipetting robot because the maximum probability of single-cell event is under 37% according to the Poisson distribution.
FACS can overcome the Poisson distribution limitation and provide an alternative method to efficiently obtain single-cell events by sorting and placing individual cells in well plates. However, the high mechanical shear stress in FACS can damage cells and affect their downstream uses. In addition, FACS is less prevalent in many laboratories due to its high machine-purchasing and operational cost.
Microfabricated devices have been utilized for capturing single cells for single cell analysis using microdroplets, dielectrophoresis, hydrodynamics, selective dewetting, mechanical techniques and microwell array on different substrates. For cell-based applications that require culturing single cells, microdroplet-based methods represent a powerful means of obtaining larger numbers of microdroplets each containing a single cell. However it is difficult to change the medium inside the microdroplets, making it not suitable for applications where the initial medium need to be replaced during experiment. In addition cells encapsulated in microdroplets are not suitable for adherent cell culture due to the lack of a substrate for cell to attach and spread.
On the other hand, trapping single cells in microwells is an attractive method to set up larger numbers of single cells for both adherent and suspension single-cell cultures due to its simplicity in device fabrication and operation as they only require physical walls and simple manipulation to load cells in compartmented spaces for subsequent culture and analysis. However, to provide a sufficient space for cell growth, the sizes of the microwells had to be made much larger (from 90-650 μm in diameter or in side length) than that of a single cell, resulting in low single-cell events (ranging from 10-30%). The decreased single-cell-loading efficiency in culture microwells is due to the inherent limitation of the Poisson distribution also seen in conventional limiting dilution method. This limitation was improved by using triangle-shaped microwells which were able to provide enlarged area for cell growth while maintain good single-cell loading efficiency (up to about 58%). However the enlarged area (about 3.5-6 times of that of a single cell) in a microwell was insufficient for cell growth beyond two days. There is still a lack of simple yet high-throughput method and device to perform single cell culture experiment.